Extraction of cycloheximide



Patented Sept. 30, A1952 lByronA E..Lea.ch, Kalamazoo, Mich., assignerto The Upjohn .,Company, Kalamazoo, Mich., a

y .corporation ofMichigan Application February-frz, 1947. ASerial No.729,201

Claims. (Cl. E60-'281) This invention relates to'a new andusefulbiosynthetic antimicrobial material, and is more ,particularlyconcerned with a method for the 2-hydroxyethyllfglutarimide. having .thefollowing structural iormulm isolation ofthe'antibiotic materialfroma'fer- (fH il) 1 mentation product containing `the saine.l 5 CH-'C 10H1-A`*It isknown that an-antibiotic can Ibe obtained HRC \.C. CH..CB,.HC Hironia Streptomycesoriseus lfermented medium, C ;GE f H v CH C/ forexample, one `which comprises glucose, meat extract, peptone, and`sodium chloride. This CH! antibiotic' is characterisedy 'SWaterSolubility 10 according to.Kornfeld,....Iones and Parke, J.

other ,insolubility chloroform insolubility, and

'acid instability (warmen veta1fero. soc. B101.

Meo.i55,-6 :(1940) and'Proc. stanMayo Clinic is, 53': 4194er); vbyiishigh activity' toward vBacillus subtilis, Staphylococcus aureaus, and

Escherichia coli; and I.by its low activity toward Saccharomycescereisiae,.Cr1/ptococcus neojor- 'mans'. -and'Rhodotoru'la sp; `Itisfurther `characterised by"its.optlcal rotation [al D2@ -1'00degrecs,vv an empirical formula 'oijCziHanN'zOiz andv byv presencein the.molecule oi three "basic groups, twosof whichare thoughtto be a part ofa guanidine'type `structure-and the third amore simple amine structure."I'his `antibiotic has "been designated streptomycin` and its structuralformula is postulatedatpages 56 and 57' of the ,book "streptomycin:.Nature and Practical Aplplications, edited'by'Selman A. Waksman,:Baltimore, The Williams and' VVllkins Co., 1949.

Ithasbeenfound that, in addition Ito ,strep- .tomycin, .there isApresent in a Sreptomyces ariseus fermented medium, such as .one.comprisingglucose, meat extract, peptoneJ and sodium chloride. anotherantibiotic substance which is characterised by "its water solubility,vether solubility, chloroform solubility, and `acid stability (Whifien.Bohonos, and Emerson, J. Bact. 52 610 `(1946i). "This antibiotic Iis'further characterised lby its low activity towardv ,Bacillus ysab'-'tllis, Staphylococcus aureaus, .and Escherichia coli, `as well as byits high activitytoward 'Saccharomyces cerevisiae, Cryptococcusneoformans, land 'Rhodotorula glutnis 2527. 'It 'is ystill .furthercharacterised by its voptical rotation la] Das 2.8 degrees, a .meltingpoint .oi 11S-11.6 degrees .centigrade, and thefact that it is a neutralsubstance anda. ketone. .Thisantibiotic has .been ydesignated.CycloheidmideWand it yhas been-shown to have an yen'niirical.formulayoi GisHzsNOi and to .the :beta-2i3edimethyle2oxocyclohexyl streptomycinfrom .beers containing .the same have, until fthe present invention,prevented .the isolation ofthe cycloheximide from fermentation productscontaining the two antibiotics. .No .method' has .been .at-hand .whereby.both of the antibiotics could :be separated v:from ya Streptomycesigriseus ybeer containing fthesame.

It is, therefore, an .object `of the ypresent invention to provide a:method Vwhereby Athe isolation of both `strep.tomycinand thecycloheximide, from fermentation vproducts containing thesame, vmay be.accomplishedswithout interference. Another object of the inventioniszthe provision @of aprocess whereby .the` cycloheximide maybeisolatedinhigh yields 'from byproducts of usual :streptomycin lextractionprocesses. A further .object vof the invention is the provision of .laproc- Iess whereby Ithe cycloheximide may be yextracted vfrom the illter'cakeordinarily discarded'in o. vstreptomycin extraction process. Abroader object of the invention is the provision oi a process `wherebythe cycloheximide may `be extracted .from a lStrcptomycE's 'griseus:fermentation 'beer containing the same. Anotherobjectil'of'thefinvention-isthe provision of :such -a process whereinvthe isolation of thei cycloheximide 4is Aaccomplished from an acidicsolution. An additional object of the invention isthe provision of aprocess whereby the 'cycloheximide may be absorbed on activated carbonfrom an acid solution. A further object of the invention is theprovision of a process whereby the cycloheximide may be adsorbed onactivated carbon from an acid solution, and 'thereafter eluted by atleast one member Yof the group consisting of water-miscible alcohols andketones. the invention is the provisionof a process for the recovery'.-of -thefcycloheximide in a` crystalline form."""'A stillfurther objectof the invention is the provision of such a process wherein the solventemployed for the crystallisation of the cycloheximide is a member of thegroup consisting of cyclohexene, amyl acetate, ethyl acetate,

will become apparent hereinafter.

Still a further object of i degrees, forms in conventional manner anacetate M; P. 143-145 degrees centigrade, an oxime M. P.

- chloroform-petroleum ether, and Yethyl ether`-- petroleum ether. Otherobjects of the invention According to usual procedure employed for the..f

isolation of streptomycin, the filtrate obtained when a S. griseusfermented medium isclaried and ltered from mycelium is further treatedto gvestreptomycin, while the filter cake is discardedas being ofnegligible value. VThis is customarily the first step in the isolationof streptomycin, and no consideration has been given to the possibilitythat the filter caire might have any value whatsoever. It has now beenfound that, according. to the-method of the present invention; theusually discarded filter cake from a streptomycin clarification is anexcellent source of cycloheximide.

The process of this invention consists essentially in the treatment ofan acidic liquid S.

griseus fermentationproduct with activated carbon and elutionof theactive material therefrom.

from the treatment of .a S. griseus fermentation beer vv'ithdecolorisingcarbon (such as Nuchar C-.190Nu).!anda diatomaceous earth filter aid(suchf-as Celite), and then lfiltering the mixture.

The lter cake is treatedLWith an alcohol or ket'onalwhich issubstantially completely watermiscible, to yeluate the cycloheximidetherefrom. Such Water-miscible alcohols .or ketonesinclude acetone, lmethyl ethylv ketone, diethylv ketone,

methanol, ethanol, propanol, isopropyl alcohol, butyl alcohol, and thelike.A The volatile solvent vis removed .from the eluate in vacuo andthe aqueousiresidue is extractedy with a chlorinated hydrocarbonsolvent, preferably'one. which boils below 4about 100 degreescentigrade. Various chlorinatedgethylenes and methanes are` especiallysuitable, and chloroform is preferred. The chloroform or other extractAis decolorised with a decolorising carbon and the chloroformremoved,whereupon the cycloheximide remains. In some instances thecycloheximideA may crystallise im.- mediately, but it isusually..advantageously/further purified as, for example, by acounter-current distribution between benzene and water (Craig, J. Biol;Chem. 155, 519 (1944)) before,v

crystallisationfrom a solvent. Amyl acetate, cyclohexene, petroleumether, ethyl acetate, ethyl ether-petroleum ether, andchloroform-petroleum ether mixtures are especially suitable vsolventsfor the crystallisation of cycloheximide. pure, `the cycloheximide meltsat 115-116 degrees centigrade, and may becrystallised from water, Abutwith crude preparations this is not the case. While the cycloheximidemay be obtained from acidic streptomycin beers by adsorption Fon acti'-When \ This is readily .accomplished inl commercial operation .by.employingthe` lter calze obtained 203-204 degrees centigrade, aldisemicarbazone 'K M. P. 1;'*76-1'78 degrees centigrade. The bacterialspectra of the 'highly-purified cycloheximide is very' similar to `thatpreviously reported by rWhiffen, lBohonos, and Emerson for less puriedmaterial, the difference being solely in degree of activity.

The following examples are illustrative only, and vare not to beconstrued as limiting the in- I' EXAMPLEs Y VPure cycloheximidei'sassigned an activityof 1000 micrograms per milligram. Assay ofproducts containing Vthe cycloheximide is bythe paper'disc plate methodof Loo,-Srkell, .'l hornberry, Ehrlich. McGuire, Savage. and SylvestenJ.Bact. 50.301

(.1945), using Saccharomyces pastoridnus y2336 as the test organism. Y f

The type of acid oracids which may in the Vfermentation beer is ofnosignificance..

The pH of the liquid fromwhich adsorption iis accomplished is likewiseunimportant, it f is merely necessary that it be not alkaline.,lg'gxivynpl'e1'` L Y.

v:vwo hundred and twenty-meinem of acidic beer from a commercialstreptomycin vfeimentx'ation was extractedV thrice with chloroform(22,11,

L and 11 liters, respectively). .Thecombined'chlo reform extracts wereevaporated to dryness, tli'e residue taken up in benzene andlyophylized. Yield, 8.3 grams assaying 830 ,micrograms/of thecycloheximide per milligram. Example 2 Four hundred andv twenty litersofacidic beer from a streptomycin vfermentation assayingj() micrograms permilliliter, or 2.1.0 grams, ofthe cycloheximide was' claried with'f1050`grams (0.25 per cent) ofl decolorising carbonlNuchar C-lSONu) and 4200'grams"(11'per cent) o fa diatomaceo'us earth lter aid (Celite). Thistreatment completely removed the cycloheximide from the solution, and'the ltrate was subjected to lfurther treatment for the separation' ofstrep,- tomycin. Portionwise elution of thel carbon"-fllter-aid-mycelium cake with acetone (46 liters total) gave 18.7 gramsof theA cycloheximide'in .they eluate, as calculated from an assay. of aportion ,Y of the total eluate. The eluate was concentrated in vacuo tolan aqueous residue having a volume of '7.6 liters and apHof 2.8. Theaqueous residue was extracted withfthreejone-'liter portions ofchloroform,`where,after the' combined extracts were treated with 285grams of decolorising carbon to give an almost colorless solution.Evaporation of the chloroform 'gave 18.9 grams ofv an. 011 which could.becrystanised directly'.

Crystallisations from 1.a' chloroform-petroleum .maracas ether mixtureyielded v9.6 r'grams"'o'fthe cycloheximide (45.7 per cent. calculatedIfrom the iamountlpresent in the original beer)`.,After A threecrystallisations, the, melting point was wctivated-adsorptive carbon,eluting fthe adsorbed cycloheximide therefrom with at least.' one" 'sol-115-'116 degrees centigrade. This material also -5 vvent off the groupyconsistingofwateremisoible crystallised satisfactorily from amylacetate, .alcohols and ketones, removing the veluting .liqcycmhexene,and ethy1 acetate, uid,v dissolving the crude cycloheximide-contain-Emmpz'e 3 ing .residuevr'in water and extracting thefcyclov A f heximidefrom the aqueous'solutionwith a chio- Five hundred'and five liters'ofacidic beer from 1'0 rinated hydrocarbon solvent, andv .crystallising astreptomycinfermentation were treated with thecyclOheXimide from asolventftherefor.. 40.25 per cent carbon and 1 percent filter aid as 3.A process as defined in claim-.12in which'tlie in Example 2. Thefiltrate was 'further 'treated Awater-niiscible eluting solventfismethanol. Y to recover streptomycin, while the filter cake .was 4. Aprocess as defined in claim 2 in whichfthe. eluted with methanol. Themethanol eluate" was Waterf-miscible eluting solvent is acetone. thenrconcentrated in vacuo 'and' treated in 5. A process as defined in claim2 in which the Example 2. The yield of materialiobtained aftercrystallization of cycloheximide is effected in a evaporation ofchloroform was 10.0 `grams assay- .solvent of the group consisting of-cyclohexena ing` 54'0 micrograms of the vcycloheximide per ethylacetate, amyl acetate, petroleum ether, milligram. mixtures ofchloroform and petroleum ether, and

Example 4 mixtures of ethyl ether and petroleum ether.

6. A process as dened in claim 2 in which the Nine hundred eightymilligrams 0f Crude eluting solvent is a member of the groupconsistcycloheximide was dissolved in milliliters of lng of acetone,methyl ethyl ketone, diethyl benzene and subjected t0 a 12D1ate0011111121'- 25 ketone, methanol, ethanol, propanol, isopropanol currentdistribution in separatory funnels, using and butanol.

40 milliliters of water as the traveling phase ao- 7. A process asdefined in claim 2 in which the cording to the method of Craig, J- B101.Chemchlorinated hydrocarbon solvent is chloroform. 155, 519 (1944). Thedistribution coefiicient is 8. A process for the recovery ofcycloheximide about 0.7, and the following table gives the re- 30 froman aqueous culture of the organism Strepsult of such an extraction.tomyces grseus, which comprises acidifying the No. 1,12%: Assay Remarksgg 2s ont an dilution.

gg s cateo dilution.

83 36 active at 300 dilution.

S0 136 active at 3,000 dilution. Main active band.

gg 164 active at 3,000 dilution.

gg 80 active at 1,000 dilution.

g8 212 active at 30 dilution.

The main active fractions can be crystalls'ed culture to dissolve andretain ,in the liquid a subby dissolving in ether or chloroform afterrestantial proportion of the streptomycin, adding moval of the benzenetherefrom, and thereafter activated adsorptive carbon to the acidiiiedculpouring the solution into a large volume of hot ture and subjectingit to absorptive treatment petroleum ether or other crystallisationsolvent. therewith, separating the solid mycelial growth The resultingmilky oil crystallises after thorough together with the activated carbonyfrom theL rubbing. Crystals may also be obtained by dismain portion ofthe liquid, eluting the adsorbed solving the product in hot cyclohexene,arnyl cycloheximide from the activated carbon' oonacetate, ethylacetate, and mixtures of petroleum tained in the separated solids byextraction of ,ether with chloroform or ethyl ether. The purer thesolids with at least one solvent of the group preparation, aftercrystallisation from any of consisting of water-miscible alcohols andketones the above solvents, can be satisfactorily recrysand recoveringcycloheximide from the elute. taliised from hot Water. 9. A process forthe recovery of cycloheximide Variations may be made without departingfrom an aqueous culture of the organism Strepfrom the spirit or scope ofthe-invention, and 60 tomyces grz'scus, which comprises acidifying theit is to be understood that I limit myself only culture to dissolve andretain in the liquid a as defined in the appended claims. substantialproportion of the streptomycin, add- I claim: ing activated adsorptivecarbon to the acidifled 1. A process for the recovery of cycloheximideculture and subjecting it to adsorptive treatment from an acidicShieptomyces griseusfermentatherewith, separating the solid mycelia1growth tion beer, which comprises treating the beer with together Withthe activated carbon from the activated adsorptive carbon, eluting theadsorbed main portion of the liquid, eluting the adsorbed cycloheximidetherefrom with at least one solcycloheximide from the activated carbonconvent of the group consisting of water-miscible tained in theseparated solids by extraction of the alcohols and ketones, removing theeluting liqsolids with acetone, and recovering cyclohexiuid, dissolvingthe cycloheximide-containing mide from the eluate by evaporation of theaceresidue in water and extracting the cyclohexitone and extraction ofthe residue with chloromide from the aqueous solution with a chloform.rinated hydrocarbon solvent. 10. A process for the recovery ofcycloheximide 2. A process for the recovery of cycloheximide from anaqueous culture of the organism Strgp.

-tomyces griseus, which comprises acidifying the `culturerliquid to`dissolve `and retain therein a substantial proportion of thestreptomycin contained,4 therein, adding activated adsorptive carbontothe acidied culture liquid and subjecting 5 itl to adsorptivetreatment therewith, separating the carbon from the liquid and elutingthe cyclo- Al'ieximide adsorbed thereon with at least one solvent of thegroup consisting of water-miscible alcohols and ketones, and recoveringcycloheximide from the eluate by removing the watermiscible solvent andextracting the residual aqueous solution with a chlorinated hydrocarbonsolvent.

. BYRON E. LEACH.

REFERENCES CITED "The following references are-01' recordv in the filevof this patent:

OTHER REFERENCES Waksman in JyBact. 51 p. 753-759 recd for publicationFeb. 1946.

Carter in J. Biol. Chem. vol. l6 0 p. 337-342 Schatz in Proc. Soc. Exp.Biol. Med. Vol. 57p. 244-2473k (1944). Y

Waksrnan et alt J. Am. Pharm. Assoc.

la (Scientific Edition). Vol. 34, page 276 (Nov. 1945).

Whi'en'et al: J. Bact. vol. 52, p. 610 (1946). Shriner and Fuson:Identication of .Or-

' ganic Compounds (JohnV lWiley; New York;

1940). 2nd Ed., pages 142, 167, 221, and 222.

1. A PROCESS FOR THE RECOVERY OF CYCLOHEXIMIDE FROM AN ACIDICSTREPTOMYCES GRISEUS FERMENTATION BEER, WHICH COMPRISES TREATING THEBEER WITH ACTIVATED ADSORPTIVE CARBON, ELUTING THE ADSORBEDCYCLOHEXIMIDE THEREFROM WITH AT LEAST ONE SOLVENT OF THE GROUPCONSISTING OF WATER-MISCIBLE ALCOHOLS AND KETONES, REMOVING THE ELUTINGLIQUID, DISSOLVING THE CYCLOHEXIMIDE-CONTAINING RESIDUE IN WATER ANDEXTRACTING THE CYCLOHEXIMIDE FROM THE AQUEOUS SOLUTION WITH ACHLORINATED HYDROCARBON SOLVENT.